Fiber Type Specific Presence of GIutathione Transferase Isoenzymes in the Rat SkeIetaI Musele Tissue

Glutathione transferases (GST) are a group of enzymes, majorly responsible for biochemical detoxification by conjugating glutathione to a set of hydrophobic ligands. The tissue isoenzyme pattern of the enzymes has been well illustrated with their significance of histological localization. But in the cases of muscle tissue, the GST isoenzyme pattern has not yet been clearly studied. In the present experiment, we have carried out an immunohistochemical analysis on the distribution of GST isoenzymes using anti GST-P and anti GST-L antibodies on cardiac, smooth and skeletal muscle tissues. The results showed that the intestinal smooth muscle and cardiac muscles are very weakly immunostained for both anti GST-L and P antibodies. In contrast, major bundles of skeletal muscles were positively responsive to GST-L antibody. Therefore, we compared the expression of GST-L in the muscle tissues of the soleus and plantaris, which were composed dominantly of type I fiber and type II fiber, respectively. The data indicated that only type II fibers in the plantaris muscle tissue were positive to GST-L antibody, which was confirmed by specific ATPase staining. And the soleus muscle, consisting mainly of type I fibers, contains a higher amount of GST-P isoenzyme than the plantaris muscle. Therefore, it can be suggested that the expression of GST isoenzyme can be used as a type-specific marker for the type II fiber of skeletal muscles. And moreover, the differential pattern of GST isoenzymes in those muscle tissues according to fiber types may contribute to explaining the differences in fatigue-sensitivity of muscles to exercise

Expression of Osteocalcin and Transglutaminase and Labelling of Bromodeoxyuridine during Fracture Healing in the Rat Tibia

The expression of osteocalcin and transglutaminase C(TGase C) during fracture healing was inwstigated with immunohistochemical studies. A transverse osteotomy was made at the proximal tibia in Sprague-Dawley male rats and immobilized with a small external skeletal fixator. The animals lU!l'e sacrificed serially I, 3, 5, 7, 14, 42 days respectively after fracture. Longitudinal sections of the healing bone were stained with pohclonal antibody against osteocalcin and TGase C, and monoclonal antibody against bromodeoxyuridine. During the intramembranous bone formation at the periosteum around the fracture site, osteocalcin was strongly expressed in the proliferating osteoprogenitor cells from the 1st day of fracture, and then, in osteoblasts, osteoid matrix and osteocytes. The expression of TGase C was weakly positive in both osteoprogenitor cells and osteoblasts. Ai the site of endochondral bone formation, which was first reoealed 5 days after fracture, cell proliferation occurred at the periphery of cartilaginous callus where the number of cells stained with BrdU was highest During the maturation of callus, those cells uere entrapped in the chondroid matrix and became larger and larger. Osteocalcin was demonstrated in the cytoplasm of chondrocytes, while chondroid matrix was negatiwly stained. TGase C was found in the cytoplasm of more centrally located and matured chondrocytes as compared with osteocalcin. Osteoid matrix was stained with osteocalcin but not with TGase C. These finding may suggest that osteocalcin participates in the early phase of endochondral bone formation, while TGase C participates in the late phase, suggesting the role of TGase C in matrix stabilization. But the reason for the difference in the expression of TGase C between the endochondral bone formation and intramembranous bone formation should be further inwstigated. Healing of IAA2Il immobilized fracture in this study was predominantly induced by intramembranous ossification rather than endochondral ossification. Periosteal osteoprogenitor cells appeared to initiate and to lead bone formation after osteotomy. These findings indicate that preservation of the periosteum is essential to achieve successful fracture healing

Purification of Sulfhydryl Oxidase from Human Foreskin Tissue and Immunohistochemical Localization

Human sulfhydryl oxidase, catalyzing the conversion of either free or bound thiol to disulfide compound, was isolated from human skin tissue to apparent homogeneity through multiple steps of ammonium sulfate salting-out, DEAE-cellulose chromatography, CM-cellulose chromatography and ACA54 gel filtration. The enzyme was shown to have a molecular weight of 65 kDa and a specific activity of 8.39 x 103 Ufmg protein. The specific polyclonal antibody was raised, with which the tissue distribution of the enzyme was studied immunohistochemically. The enzyme is present ubiquitously in most human tissues. However, the granular layer of the epidermis, stromal tissues of the breast and uterine cervix, hepatocytes and islets of the pancreas are noted to contain a comparatively high amount of the enzyme

Monoclonal Antibodies to Human Transglutaminase 4

Transglutaminase 4 (TG4) is a member of the enzyme family that catalyzes the calcium-dependent post-translational modification of proteins via cross-linking, polyamination, or deamidation. TG4 exhibits prostate-specific expression pattern and plays a crucial role in the formation of the copulatory plug in rodents. However, the physiological function(s) of human TG4 remains speculative. Human TG4 has been postulated to participate in the maturation process of sperm by modifying its cell surface, which results in suppression of sperm antigenicity in the female genital tract. To better understand the pathophysiological role of TG4 in prostate tissue, we generated monoclonal antibodies (MAb) against human TG4 in mice by repeated injections with the recombinant human TG4. Western blot analysis demonstrated that the selected MAbs react specifically with TG4, but not with other isoenzymes of the TG family. Immunocytochemical and immunohistochemical analyses showed that specific staining is observed with the cells overexpressing TG4 and with the paraffin-embedded prostate tissue specimens obtained from the benign prostate hyperplasia and prostate cancer patients, respectively. Our results indicate that these MAbs are suitable for detecting TG4 in the cultured cells or prostate tissues for investigating the biological functions of human TG4.Shin DM, 2004, J BIOL CHEM, V279, P15032, DOI 10.1074/jbc.M308734200Jeon JH, 2003, EMBO J, V22, P5273Lorand L, 2003, NAT REV MOL CELL BIO, V4, P140, DOI 10.1038/nrm1014Jeon JH, 2002, BIOCHEM BIOPH RES CO, V294, P818An G, 1999, UROLOGY, V54, P1105Dubbink HJ, 1999, GENE, V240, P261Dubbink HJ, 1999, LAB INVEST, V79, P141Choi K, 1998, EXP MOL MED, V30, P41Esposito C, 1996, J BIOL CHEM, V271, P27416Dubbink HJ, 1996, BIOCHEM J, V315, P901SEITZ J, 1991, BIOCHIM BIOPHYS ACTA, V1078, P139PAONESSA G, 1984, SCIENCE, V226, P852MUKHERJEE DC, 1983, SCIENCE, V219, P989WILLIAMSASHMAN HG, 1977, BIOCHEM BIOPH RES CO, V79, P1192WILLIAMS.HG, 1972, P NATL ACAD SCI USA, V69, P2322

Hepatoprotective and Antioxidative Activities of Cornus officinalis against Acetaminophen-Induced Hepatotoxicity in Mice

The fruit of Cornus officinalis Sieb. et Zucc. is commonly prescribed in Asian countries as a tonic formula. In this study, the hepatoprotective effect of ethanolic extracts of the fruit of C. officinalis (ECO) was investigated in a mouse model of acetaminophen- (APAP-) induced liver injury. Pretreatment of mice with ECO (100, 250, and 500 mg/kg for 7 days) significantly prevented the APAP (200 mg/kg) induced hepatic damage as indicated by the serum marker enzymes (AST, ALT, and LDH). Parallel to these changes, ECO treatment also prevented APAP-induced oxidative stress in the mice liver by inhibiting lipid peroxidation (MDA) and restoring the levels of antioxidant enzymes (SOD, CAT, and HO-1) and glutathione. Liver injury and collagen accumulation were assessed using histological studies by hematoxylin and eosin staining. Our results indicate that ECO can prevent hepatic injuries associated with APAP-induced hepatotoxicity by preventing or alleviating oxidative stress